CERAMIDE, A SECOND MESSENGER
In recent years the importance of ceramide as second messenger in signal transduction has been recognized. It has become clear that the signalling induced by a number of cytokines is mediated by changes in the intracellular concentration of this lipid [1,2]. For example, crucial for the transduction of the signal exerted by TNF-.alpha. (tumor necrosis factor alpha) upon binding to its receptor are local changes in ceramide concentration in specific regions, or invaginations, of the plasma membrane. Upon binding of the cytokine to its receptor, a sphingomyelinase catalyzes the conversion of sphingomyelin into phosphorylcholine and ceramide. The ceramide that is generated in this manner propagates the signal by activating specific protein kinases and phosphatases, resulting in a cellular response. FIG. 1 gives an overview of the signalling mechanism of TNF-.alpha. and other cytokines such as interferon gamma and interleukin 6.
There is now convincing experimental evidence for the role of ceramide in signalling. It has been shown that the effects of TNF-.alpha. can be experimentally mimicked by administration of a permeable ceramide with truncated fatty acyl moiety or, alternatively, by the generation of ceramide at the cell surface by the treatment of cells with a bacterial sphingomyelinase (see e.g. ref. 2).
The above described signal transduction process is most likely a highly local event, occurring near the cytokine receptor. The concentration of ceramide in the plasma membrane is believed to be very low under normal conditions. However, considerable amounts of ceramide are present in the plasma membrane as a building block in sphingomyelin. The hydrolysis of sphingomyelin would allow a considerable local change in ceramide concentration and subsequent signal propagation.
Via action of a specific transferase, ceramide can be reconverted to sphingomyelin by transfer of the phosphorylcholine moiety from phosphatidylcholine (PC), resulting in the concomitant formation of diacylglycerol. The total pathway, resulting in the netto hydrolysis of phosphatidylcholine to phosphorylcholine and diacylglycerol, is named the sphingomyelin cycle [2].
CERAMIDE AND SPHINGOLIPID METABOLISM
Obviously, not all fluctuations in intracellular ceramide concentrations are affecting signal transduction. Ceramide is extensively metabolized in cells. The lipid is synthesized at the membrane of the endoplasmic reticulum from acylCoA and sphingosine. It may be converted at the level of the Golgi apparatus into sphingomyelin, glucosylceramide and related complex gangliosides, or galactosylceramide and related globosides and sulfatides. Sphingomnyelin and glycosphingolipids are also catabolized into ceramide and other components in the lysosomal compartment of cells. The intralysosomally formed ceramide may be locally hydrolyzed into sphingosine and fatty acid by the action of the lysosomal ceramidase or it may be exported to the cytosol and re-used for synthesis of sphingolipids. A schematic overview of the ceramide metabolism is presented in FIG. 2.
SPHINGOLIPIDOSES: GAUCHER DISEASE
In man a number of inherited disorders in lysosomal sphingolipid catabolism occur, the so called sphingolipidoses (see Table 1). For example, an inherited deficiency of the lysosomal sphingomyelinase underlies Niemann-Pick disease, and defective activity of the lysosomal ceramidase causes Farber disease. The most frequently encountered sphingolipidosis is Gaucher disease [3]. The metabolic basis of this disorder is a deficiency of the lysosomal beta-glucosidase, glucocerebrosidase (E.C.3.2.1.45). This enzyme catalyzes the hydrolysis of glucosylceramide (glucocerebroside) to glucose and ceramide. In patients with Gaucher disease glucosylceramide accumulates in tubular aggregates, in particular in lysosomes of macrophages. The lipid-laden macrophages have a typical morphology and are usually referred to as `Gaucher cells`. In the course of clinical manifestation of Gaucher disease the abnormal macrophages may accumulate in large quantities in various body locations, such as the bone marrow compartment, spleen, liver, kidney, and lungs. The most pronounced clinical symptoms associated with Gaucher disease are progressive splenomegaly, hepatomegaly, and skeletal deterioration. Most Gaucher disease patients do not develop neurological complications. The common non-neuronopathic form of the disease is called Type 1 Gaucher disease. In very severe cases of Gaucher disease characteristic neurological abnormalities may also occur, resulting in lethal complications at infantile (Type 2) or juvenile (Type 3) age [3].
GAUCHER CELLS
The glucosylceramide-laden Gaucher cells are believed to play a crucial role in the pathophysiology. Their massive presence in various body locations is thought to lead to local pathology.
Gaucher cells should not be viewed as inert containers of glycosphingolipid. The storage cells are viable and actually, being activated macrophages, secrete large amounts of specific proteins such as hydrolases and cytokines. These factors in turn act as pathogenetic agents that cause local tissue damage and turnover. Moreover, Gaucher-cell derived factors such as cytokines promote the recruitment of additional activated macrophages (see FIG. 3 for a schematic overview).
Recently a sensitive marker for Gaucher cells has been discovered by us [4]. Using the technique of in situ hybridization we observed that Gaucher cells synthesize large quantities of the secretory enzyme chitotriosidase, the human analogue of chitinases present in various species. This explains the dramatic elevation in plasma chitotriosidase levels in clinically affected Gaucher patients. On the average chitotriosidase levels are about 1000 fold higher in plasma of these patients as compared to corresponding normal subjects. In presymptomatic or asymptomatic individuals with an inherited glucocerebrosidase deficiency plasma chitotriosidase levels are (almost) within the normal range (see Table 2). Interestingly, elevated levels of plasma chitotriosidase have also been noted for patients with other sphingolipidoses, in particular Niemann-Pick disease [5].
It has been observed that in cultured macrophages, derived from peripheral blood monocytes, the concentration of glucosylceramide gradually increases. The increase in glycolipid is more pronounced when cells are grown in the presence of conduritol B-epoxide, a potent irreversible inhibitor of glucocerebrosidase. After approximately 7 days of culture the macrophages start to produce chitotriosidase mRNA and secrete the enzyme [4,6]. The expression of the chitotriosidase gene subsequently dramatically increases: after about three weeks chitotriosidase constitutes almost 1% of the total synthesized protein, as revealed by the incorporation of radioactively labeled methionine [7]. The continuous presence in the culture medium of glucosylceramide, or of conduritol B-epoxide (an irreversible inhibitor of lysosomal glucocerebrosidase), promotes chitotriosidase expression.
THERAPEUTIC INTERVENTION FOR GAUCHER DISEASE
The sparse and anecdotal information on the natural history of Gaucher disease indicates that although clinical symptoms develop progressively, the disease manifestation is usually not a gradual proces. In the case of most patients abnormalities develop rapidly at a particular age in a specific tissue, may subsequently stabilize for considerable time, to become next rapidly progressive again. In other words, disease progression has a local and chaotic feature. Most likely, Gaucher cells play a critical role in these local pathogenetic processes. The presence of the activated storage cells will locally induce tissue damage and turnover, and promote recruitment of activated macrophages at these sites, initiating a chaotic cascade of pathological events (see FIG. 3). According to this concept, a major beneficial effect should be exerted by a disruption or prevention of the pathological cascade. The various therapeutic approaches for Gaucher disease that have been considered are discussed here below.
ENZYME SUPPLEMENTATION THERAPY
For more than thirty years supplementation of macrophages of Gaucher patients with human glucocerebrosidase has been seriously considered as a therapeutic option. Efforts to develop a therapy for Gaucher disease have been largely unsuccessful for many years due to the unavailability of sufficient amounts of pure human glucocerebrosidase and the poor targeting of intravenously administered enzyme to lysosomes of tissue macrophages. Only since 1990 an effective therapeutic intervention for Gaucher disease is available that is based on the chronic supplementation of patients with human glucocerebrosidase [8]. Administered by intravenous infusion is a human glucocerebrosidase that is modified in its N-linked glycans such that mannose-residues are terminally exposed. The modification favours uptake via mannose receptors. Improved targeting of the modified (`mannose-terminated`) enzyme to lysosomes of tissue macrophages occurs via mannose-receptor mediated endocytosis. Different dosing regimens that vary with respect to total dose (15-240 U/kg body weight.month) and frequency of administration (three times weekly to biweekly) are presently used (see e.g. ref. 9). Glucocerebrosidase isolated from human placenta (Ceredase; alglucerase) and enzyme recombinantly produced in CHO-cells (Cerezyme; imiglucerase) have been found to be equally potent in reversing some of the clinical signs associated with the disease [10].
The most pronounced beneficial effects of enzyme replacement therapy are the reductions in liver and spleen volumes, and the improvements in hematological parameters such as hemoglobulin concentration and thrombocyte and leukocyte counts. Marked interindividual differences exist in the rate and extent of clinical response, even among related patients that are treated with the same dosing regimen [9]. In general, the most marked clinical improvements occur within the first year of treatment, accompanied by a pronounced correction of biochemical serum abnormalities. A complete reversal of clinical signs and complete normalization of serum abnormalities, such as elevated levels of angiotensin converting enzyme, tartrate-resistant acid phosphatase and chitotriosidase, is not accomplished by enzyme therapy, not even in the case of patients that receive a high dose of glucocerebrosidase for a number of years [11]. The partial correction following enzyme therapy is in contrast to the complete correction that is noted for patients that underwent a successful bone marrow transplantation.
Conflicting views still exist with respect to the optimal dosing regimen for enzyme therapy. Whereas low dosing regimens may be (almost) equally successful to high dosing regimens in generating hematological improvements, this is still questionable with respect to intervention of the bone disease.
Presently more than 1500 patients are receiving enzyme therapy. This recent development has attracted considerable scientific and public attention, also due to the high costs and potential risks that are involved. The costs associated with successful therapy have hitherto been exceptionally high ($100,000 to $400,000 annually per patient); leading to the belief that the enzyme therapy of Gaucher disease is the most expensive drug treatment for any disease. Although the alglucerase preparation is known to contain minor amounts of HCG and other impurities, the experience so far indicates that enzyme therapy is safe.
The enzyme therapy for Gaucher disease is considered to be a model case for the future development of treatments for other rare genetic disorders--a point perhaps best illustrated by the organisation in February/March 1995 of a Technology Assessment Conference at the National Institutes of Health, Bethesda, USA, that was specifically devoted to Gaucher disease. This type of conference is only organised when there is an exceptionally pressing health care need. During the conduct of the conference, a panel of 12 independent experts took evidence from leading scientists and clinicians in the field of Gaucher disease; the panel concluded that enzyme therapy is effective in reversing a number of clinical signs associated with Gaucher disease. Furthermore, it was stressed that reduction of the costs and the associated potential risks of human protein replacement therapy are critical issues both from the point of view of patient care and health care economics [12].
OTHER THERAPEUTIC APPROACHES
A successful treatment of Gaucher disease by bone marrow transplantation has been accomplished for a limited number of juvenile Gaucher patients. The introduction of the normal genetic information for glucocerebrosidase in hematopoietic stem cells results in the formation of blood cells that are able to hydrolyze glucosylceramide at normal rates. The fact that clinical abnormalities disappear in Gaucher patients following a successful bone marrow transplantation indicates that the presence of blood cells with normal glucocerebrosidase activity is sufficient for prevention of disease symptoms. Unfortunately, the applicability of bone marrow transplantation as treatment for Gaucher disease is quite restricted due to the limited availability of bone marrow from matched donors and the considerable morbidity associated with this intervention, particularly in the case of adults.
In recent years the option of gene therapy of Gaucher disease is intensively studied. In general, the following approach is envisioned. Pluripotent hematopoietic stem cells are isolated and transduced with a vector containing human glucocerebrosidase cDNA. After successful transduction the stem cells are re-introduced in the patient. Although data obtained with animal studies suggest that Gaucher disease is an attractive candidate for gene therapy, a number of serious problems still have to be solved before efficient intervention in this manner can be expected. A major disadvantage is that the `genetically corrected` stem cells and their progeny most likely have no selective advantage. It is therefore assumed that in order to be effective gene therapy has to result in a stable correction of a major proportion of the pluripotent stem cells. For a critical evaluation of the state of the art concerning gene therapy see ref. 13.
A distinct therapeutic approach that has been proposed for Gaucher disease is the so called `substrate deprevation therapy` [14-16]. It is argued that a marked reduction of the synthesis of glucosylceramide may have a beneficial effect because the amount of glucosylceramide that has to be degraded by macrophages would be lower. Several inhibitors of glucosylceramide synthase have been developed, e.g. 1-phenyl-decanoylamino-3-morpholino-1-propanol (PDMP) and its analogue 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) [14], butyl-deoxynojirimycin [15] and butyl-deoxygalactonojirimycin [16].
A disadvantage of the `substrate deprevation` approach is that a priori not only the synthesis of glucosylceramide but also that of more complex glycosphingolipids is inhibited. Moreover, the presently available inhibitors of glucosylceramide synthase are known to exert a number of important biological effects that may limit their applicability as therapeutic agent. For example, PDMP is known to induce apoptosis in some cell types. Butyl-deoxynojirimycin is known to inhibit also the lysosomal glucocerebrosidase and the a-glucosidase I, an ER enzyme that plays a critical role in trimming of N-linked glycans in newly formed glycoproteins and as such in quality control of protein folding. The antiviral action of butyl-deoxynojirimycin is thought to be caused by its inhibitory effect on glycoprotein modification. Moreover, it was recently reported that glucosylceramide synthase inhibitors induce the synthesis of the enzyme. Consequently, these inhibitors would need to be chronically administered to Gaucher patients since their withdrawal would be followed by an abnormally high level of glucosylceramide synthase activity and increased load on glucosylceramide [14].